Controlled and Localized Release of Retinoids to Improve Neointimal Hyperplasia

ABSTRACT

Controlled release vascular implants, such as vascular grafts, stents, wraps, and gels comprising a biocompatible polymer and all trans retinoic acid (ATRA), or its derivatives, can be used to treat, prevent, or inhibit thrombosis and/or neointimal hyperplasia which may otherwise be induced by prosthetic implantation. In particular, the implants herein can inhibit smooth muscle cell proliferation, neointimal hyperplasia, and upregulate antithrombotic genes and nitric oxide production in the vasculature. Further, the implants are capable of delivering controlled and predictable localized concentrations of ATRA.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/546,033, filed Nov. 18, 2014, now allowed, which is a continuation ofU.S. patent Application Ser. No. 12/497,187, filed Jul. 2, 2009, nowabandoned, which claims the benefit of the filing date of U.S.Provisional Application Ser. No. 61/077,949 filed Jul. 3, 2008, each ofwhich is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present disclosure relates to vascular implants incorporating alltrans-retinoic acid or its derivatives reduce thrombosis and/orneointimal hyperplasia following surgical implantation.

BACKGROUND OF THE INVENTION

Atherosclerosis is prevalent in all developed nations and is the leadingcause of death and disability in the United States. A debilitating anddisabling sequela of atherosclerosis is peripheral arterial disease(PAD). Persons with PAD often have impaired function and quality oflife, regardless of symptoms. For those with severe PAD, often lowerextremity bypass grafting remains the only option for limb salvage.

The gold standard conduit for infrainguinal bypass grafting isautologous vein. While the patency for infrainguinal vein grafts remainsapproximately 70% at 5 years, vein is not available in approximatelyone-third of patients due to intrinsic venous disease or prior veinharvesting. In these cases, expanded polytetrafluoroethylene (ePTFE)grafts are the most commonly used alternative bypass conduit. However,the primary patency rates for infrapopliteal ePTFE bypass grafts aredismal. Prosthetic bypass graft failure occurs secondary to eitherprogression of atherosclerotic disease, thrombosis, or development ofneointimal hyperplasia.

Problems associated with using prosthetic grafts are so severe thatcardiac surgeons do not use them for coronary artery bypass grafting(CABG). Patients that require CABG would benefit significantly fromoff-the-shelf prosthetic grafts as often times they do not have healthyveins or arteries to perform the procedure. Although ePTFE grafts arethe current standard for prosthetic infrainguinal bypass grafting, theycan be thrombogenic, especially when used in small diameter below-kneerevascularization procedures. Stenosis due to neointimal hyperplasiaremains a challenge to their long-term efficacy.

Several types of surface modification strategies have been utilized tochange the nature of the interaction between blood and the prostheticgraft. Most of these strategies have focused on permanently immobilizingan antithrombogenic compound or creating a protein-resistant surface,with variable results. For example, heparin has been widely used as anantithrombotic and antiproliferative agent to modify the surface ofvascular grafts in order to reduce thrombus formation and neointimalhyperplasia. In animal models, heparin-modified ePTFE graftssignificantly reduced acute thrombosis and anastomotic neointimalhyperplasia. However, the possible formation of antiplatelet antibodiesand the associated heparin-induced thrombocytopenia can result in deadlyoutcomes.

Therefore, there exists a need for improved graft modificationtechnologies which enable treatment or inhibition of neointimalhyperplasia while avoiding systemic or toxic side effects.

SUMMARY OF THE INVENTION

Controlled release vascular implants, such as vascular grafts, stents,gels, and wraps, comprising a biocompatible polymer and all transretinoic acid (ATRA), or its derivatives, can be used to treat, prevent,or inhibit thrombosis and neointimal hyperplasia which may otherwise beinduced by prosthetic implantation. In particular, the implantsdescribed herein can inhibit smooth muscle cell proliferation,neointimal hyperplasia, and upregulate antithrombotic genes and nitricoxide production in the vasculature. Further, the implants are capableof delivering controlled and predictable localized concentrations ofATRA.

In one aspect, the present disclosure provides methods for reducing orpreventing the occurrence of neointimal hyperplasia and/or thrombosisfollowing implantation of a vascular implant, said method comprising,contacting a vascular implant with all-trans retinoic acid (ATRA); andimplanting the vascular implant in a patient in need of thereof, whereinthe vascular implant comprises a biocompatible polymeric matrix; and thevascular implant releases a therapeutically effective amount of ATRAsufficient to inhibit or prevent neointimal hyperplasia when implantedin the patient.

In a second aspect, the present disclosure provides methods forpreparing a modified vascular implant, comprising, providing a vascularimplant; and contacting the vascular implant with all-trans retinoicacid (ATRA) to yield a modified vascular implant, wherein the modifiedvascular implant comprises a biocompatible polymeric matrix; and themodified vascular implant releases a therapeutically effective amount ofATRA sufficient to inhibit neointimal hyperplasia and/or thrombosis whenimplanted in a patient.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present disclosure provides methods for reducingor preventing the occurrence of neointimal hyperplasia followingimplantation of a vascular implant, said method comprising, contacting avascular implant with all-trans retinoic acid (ATRA); and implanting thevascular implant in a patient in need of thereof.

As used herein, the phrase “therapeutically effective amount” refers tothe amount of active compound or pharmaceutical agent that elicits thebiological or medicinal response that is being sought in a tissue,system, animal, individual or human by a researcher, veterinarian,medical doctor or other clinician, which includes one or more of thefollowing:

-   -   (1) preventing the disease; for example, preventing a disease,        condition or disorder in an individual who may be predisposed to        the disease, condition or disorder but does not yet experience        or display the pathology or symptomatology of the disease;    -   (2) inhibiting the disease; for example, inhibiting a disease,        condition or disorder in an individual who is experiencing or        displaying the pathology or symptomatology of the disease,        condition or disorder; and    -   (3) ameliorating the disease; for example, ameliorating a        disease, condition or disorder in an individual who is        experiencing or displaying the pathology or symptomatology of        the disease, condition or disorder (i.e., reversing the        pathology and/or symptomatology) such as decreasing the severity        of disease.

The term “biocompatible”, as used herein is intended to describematerials that do not elicit a substantial detrimental response in vivo.

As used herein, “biodegradable” polymers are polymers that fully degradeunder physiological or endosomal conditions. In preferred embodiments,the polymers and polymer biodegradation byproducts are biocompatible.Biodegradable polymers are not necessarily hydrolytically degradable andmay require enzymatic action to fully degrade.

The phrase “endosomal conditions”, as used herein, relates to the rangeof chemical (e.g., pH, ionic strength) and biochemical (e.g., enzymeconcentrations) conditions likely to be encountered within endosomalvesicles. For most endosomal vesicles, the endosomal pH ranges fromabout 5.0 to 6.5.

The phrase “physiological conditions”, as used herein, relates to therange of chemical (e.g., pH, ionic strength) and biochemical (e.g.,enzyme concentrations) conditions likely to be encountered in theintracellular and extracellular fluids of tissues. For most tissues, thephysiological pH ranges from about 7.0 to 7.4.

As used herein, the term “patient” refers to animals, including mammals,preferably humans.

In certain embodiments, the vascular implant as described above is avascular graft, a vascular stent, a wrap, or a gel. In a particularembodiment, the vascular implant is a vascular graft. In anotherparticular embodiment, the vascular implant is a vascular stent. Inanother particular embodiment, the vascular implant is a wrap, such asan anastomoses wrap. In another particular embodiment, the vascularimplant is a gel. Such wraps and gels may be placed around at least aportion of a vascular graft or stent for implantation. Thereby, avascular implant can comprises a wrap or gel comprising thebiocompatible polymeric matrix and ATRA and a vascular stent or graft,wherein the wrap or gel is placed around at least a portion of thevascular stent or graft.

In another embodiment, a vascular graft can be implanted in a patient inneed thereof according to methods known to one skilled in the art, andthen a wrap or gel comprising the biocompatible polymeric matrix andATRA can be placed around at least a portion of the implanted vasculargraft and at least a portion of the blood vessel into which the grafthas been implanted. As such, ATRA can diffuse through the vascular graftand the blood vessel into which the graft has been implanted in order toprevent and/or inhibit neointimal hyperplasia.

In another embodiment, a vascular graft which has been coated over atleast a portion of its exterior surface with a wrap or gel comprisingthe biocompatible polymeric matrix and ATRA can be implanted in apatient in need thereof according to methods known to one skilled in theart.

In another embodiment, a vascular stent can be implanted in a patient inneed thereof according to methods known to one skilled in the art, andthen a wrap or gel comprising the biocompatible polymeric matrix andATRA can be placed onto at least a portion of the interior surface ofthe implanted stent.

In another embodiment, a vascular stent which has been coated over atleast a portion of its exterior surface with a wrap or gel comprisingthe biocompatible polymeric matrix and ATRA can be implanted in apatient in need thereof according to methods known to one skilled in theart. As such, the wrap or gel can be entrapped between the exteriorsurface of the vascular stent and the interior surface of the bloodvessel into which the stent has been implanted.

The vascular implants generally comprise a biocompatible polymericmatrix. For example, the biocompatible polymeric matrix can comprise apolyester, polyurethane, polycarbonate, polyanhydride, polyphosphoester,or a mixture thereof. The biocompatible polymeric matrix can beelastomeric; or the biocompatible polymeric matrix can be a gel.

As used herein, an elastomer is a macromolecular material that canreturn rapidly to the approximate shape from which it has beensubstantially distorted by a weak stress. For example, rubber is acommon elastomer.

The term “gel” as used herein is directed to a continuousthree-dimensional crosslinked polymeric network integrating a liquidinto the interstices of the network. The crosslinked polymeric networkprovides the gel structure. Depending upon their degree of structure,gels can have a broad spectrum of properties, ranging from flowing gelswhich are slightly more viscous than water to nonflowing gels which arevery rigid. The term “flowing gels” as used herein refers to gels whichflow under the force of gravity when unconfined at ambient atmosphericconditions. “Nonflowing gels” do not flow under these conditions.

In certain embodiments, the biocompatible polymeric matrix is apolyester, such as a poly(citric acid-diol) or a poly(glycerol-diacid).

A poly(citric acid-diol), as used herein, is a polyester prepared fromcitric acid (a tri-carboxylic acid monomer), and a second monomercomprising two alcohol functional groups (a “diol”) according to methodsfamiliar to one skilled in the art. For example, suitable poly(citricacid diols) can be prepared as described in U.S. Patent ApplicationPublication Nos. 2005/0063939 and 2007/0208420, which are herebyincorporated by reference in their entirety. Examples of diols include,but are not limited to, aromatic-diols (e.g., hydroquinone, catechol,resorcinol), C₂-C₂₀ alkyl-diols, C₂-C₂₀ alkenyl-diols (e.g.,tetradeca-2,12-diene-1,14-diol), and mixtures thereof. The diols mayalso include substituents as well. Reactive groups like amines andcarboxylic acids will increase the number of sites available forcross-linking. Amino acids and other biomolecules will modify thebiological properties of the polymer. Aromatic groups, aliphatic groups,and halogen atoms will modify the inter-chain interactions within thepolymer. Diols further include macromonomer diols such as polyethyleneoxides, and N-methyldiethanoamine (MDEA).

In certain embodiments, the diol comprises one or more C₂-C₂₀alkyl-diols, C₂-C₂₀ alkenyl-diols, or mixtures thereof. In certain otherembodiments, the diol comprises one or more C₂-C₂₀ alkyl-diols, such asa C₆-C₂₀ alkyl-diol, or a C₆-C₁₄ alkyl-diol, or a C₆-C₁₂ alkyl-diol. Forexample, the diol can comprise an α,ω-C₂-C₂₀ alkanediol, such as1,12-dodecanediol, 1,10-decanediol, 1,8-octanediol, or a mixture thereofIn another example, the diol can comprise 1,10-decanediol,1,8-octanediol, or a mixture thereof. In another example, the diol cancomprise 1,8-octanediol (e.g., the polyester ispoly(1,8-octanediol-citrate).

The poly(citric acid-diol) may be crosslinked, for example, byoptionally including one or more hyperbranching monomers, such as amonomer comprising three alcohol functional groups (a “triol”), in orderto control the degradation thereof. For example, glycerol can be addedin addition to the citric acid and diol monomer (0-3 mol %, provided themolar ratio of carboxyl and hydroxyl group among the three monomers wasmaintained as 1/1). Glycerol is a hydrophilic component, and itsaddition can facilitate the water penetration into the network filmswhich results in the faster degradation rate. Increasing amounts ofglycerol can increase the break strength and Young's modulus of theresulting polyester. For example, the Young's modulus can range from 1to 16 MPa, with strengths and strains at break of up to 10 MPa and 500%,respectively. Depending on the synthesis conditions, total degradationtime may range from months to years. Degradation within 6 to 12 monthsis preferred.

A poly(glycerol-diacid), as used herein, is a polyester which isprepared from a triol monomer, glycerol, and a second monomer comprisingtwo carboxylic acid functional groups (a “diacid”) according to methodsfamiliar to one skilled in the art. For example, suitablepoly(glycerol-diacid)s can be prepared as described in U.S. PatentApplication Publication No. 2003/0118692, which is hereby incorporatedby reference in its entirety. Examples of diacids include, but are notlimited to, aromatic-diacids (e.g., terephthalic acid andcarboxyphenoxypropane), C₂-C₂₀ alkyl-diacids, C₂-C₂₀ alkenyl-diacids,and mixtures thereof. The diacids may also include substituents as well.Reactive groups like amine and hydroxyl will increase the number ofsites available for cross-linking. Amino acids and other biomoleculeswill modify the biological properties of the polymer. Aromatic groups,aliphatic groups, and halogen atoms will modify the inter-chaininteractions within the polymer.

In certain embodiments, the diacid comprises one or more C₂-C₂₀alkyl-diacids, C₂-C₂₀ alkenyl-diacids, or mixtures thereof. In certainother embodiments, the diacids comprises one or more C₂-C₂₀alkyl-diacids. For example, the diacid can comprise an α,ω-C₂-C₂₀alkanediacid, such as, sebacic acid, malonic acid, succinic acid,glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, ora mixture thereof. In another example, the diacid can comprise sebacicacid (e.g., the polyester is poly(glycerol-sebacate).

The poly(glycerol-diacid) may be crosslinked, for example, by includingone or more hyperbranching monomers, such as a monomer comprising threecarboxylic acid functional groups (a “triacid”), may be optionallyincluded in the poly(glycerol-diacid), in order to control thedegradation thereof. For example, citric acid can added in addition tothe glycerol and diacid monomers (0-3 mol %, provided the molar ratio ofcarboxyl and hydroxyl group among the three monomers was maintained as1/1).

The elastic modulus and degradation rate of the polymer is easilyadjusted by modifying the cross-link density. In certain embodiments,the cross-link density of elastomeric polymers produced according to theinvention may be 40% or less, less than 30%, less than 20%, less than10%, or less than 5%. The polymer may have a crosslink density of 40% orless, less than 30%, less than 20%, less than 20%, less than 5%, lessthan 1%, less than 0.5%, or less than 0.05%.

In one embodiment, the glycerol-diacid copolymers of the invention havea tensile elastic modulus of 5 MPa or less. One skilled in the art willrecognize that the modulus of the polymer may be adjusted depending onthe application. For example, the polymer may have a modulus less than 3MPa, less than 1 MPa, less than 0.5 MPa, less than 0.3 MPa, or less than0.1 MPa. The polymer may have a maximum elongation greater than 250%.

Catalysts may be used to reduce reaction temperature, shorten reactiontime, and increase individual chain length for preparation of thepolyesters described above. However, the catalyst should bebiocompatible or easily removed. An exemplary FDA-approved catalyst isstannous octoate (bis(2-ethylhexanoate)tin(II)).

ATRA can be incorporated into the biocompatible polymeric matrix, forexample, as microparticles or nanoparticles comprising the ATRA whichcan be embedded within the biocompatible polymeric matrix. In anotherembodiment, ATRA can be incorporated in the biocompatible polymericmatrix as microparticles and/or nanoparticles of ATRA.

Alternatively, the ATRA can be incorporated into the biocompatiblepolymeric matrix by encapsulation in micelles or liposomes, wherein themicelles or liposomes are embedded within the biocompatible polymericmatrix. In another alternative, the ATRA can be absorbed, suspended, ordissolved within the biocompatible polymeric matrix. In certainembodiments, the ATRA is suspended or dissolved within the biocompatiblepolymeric matrix. In certain other embodiments, the ATRA is absorbed bythe biocompatible polymeric matrix.

The vascular implants as described according to any of the precedingembodiments, can release a therapeutically effective amount of ATRAsufficient to inhibit or prevent neointimal hyperplasia and/orthrombosis when implanted in a patient.

In one embodiment, the ATRA can be released from the vascular implantsat a rate of about 0.001 to 5 mg per gram of polymer per day whenmeasured according to high performance liquid chromatography (in vitro).In certain embodiments, the ATRA is released from the vascular implantsat a rate of about 0.001 to 1.2 mg per gram of polymer per day; or about0.001 to 0.78 mg per gram of polymer per day; or about 0.001 to 0.58 mgper gram of polymer per day. In a particular embodiment, the ATRA isreleased from the vascular implants at a rate of about 0.001 to 0.39 mgper gram of polymer per day.

In another embodiment, the vascular implant, as defined by any one ofthe preceding embodiments, can comprise about 0.001 to 15 wt % ATRA withrespect to the biocompatible polymeric matrix. In certain embodiments,the vascular implant comprises about 0.001 to 10 wt %; or about 0.001 to7.5 wt %; or about 0.001 to 5 wt % ATRA with respect to thebiocompatible polymeric matrix. In a particular embodiment, the vascularimplant comprises about 0.001 to 3 wt % ATRA with respect to thebiocompatible polymeric matrix.

In yet another embodiment, the therapeutically effective amount of ATRAcan be released by the vascular implant, as defined by any one of thepreceding embodiments, for a period of about 1 day to about 12 weeks. Inparticular, the therapeutically effective amount of ATRA can be releasedby the vascular implant, as defined by any one of the precedingembodiments, for a period of about 1 day to 10 weeks; or about 1 day to8 weeks; or about 1 day to 6 weeks. In certain embodiments, thetherapeutically effective amount of ATRA can be released by the vascularimplant, as defined by any one of the preceding embodiments, for aperiod of about 1 day to 4 weeks.

Further, the biocompatible polymeric matrix coating comprising the ATRAin any of the preceding embodiments can have a thickness ranging fromabout 0.01 to 3 mm; or about 0.1 to 3 mm. In certain embodiments, thebiocompatible polymeric matrix coating comprising the ATRA in any of thepreceding embodiments can have a thickness ranging from about 1 to 10 μmor 2 to 5 μm.

In one embodiment of the first aspect, the present disclosure providesmethods for reducing or preventing the occurrence of neointimalhyperplasia following implantation of a vascular implant as describedaccording to any of the preceding embodiments.

ATRA

All-trans retinoic acid (ATRA) is a hydrophobic, lipid- andethanol-soluble compound which, in vitro, has demonstrated a multitudeof vasoprotective properties, including inhibition of vascular smoothmuscle cell (VSMC) migration, VSMC proliferation, and extracellularmatrix production (see, for example, Miano et al. Circulation 1996 May15; 93(10):1886-95; and Johst U, et al. J Cardiovasc Pharmacol 2003April; 41(4):526-35.) ATRA stimulates VSMC apoptosis (see, for example,Orlandi, A. et al. Arterioscler Thromb Vasc Biol 2005 February;25(2):348-53; and Orlandi, A. et al., Arterioscler Thromb Vasc Biol 2001July; 21(7):1118-23). With respect to endothelial cells, ATRA has beenshown to modulate endothelial cell growth and phenotype (see, forexample, Braunhut, S. J. et al., Microvasc Res 1991 January;41(1):47-62;and Gaetano, C. et al., Circ Res 2001 Mar. 2;88(4):E38-E47). Further,ATRA has been shown to increase nitric oxide synthesis andthrombomodulin release from endothelial cells (see, for example, Achan,V. et al., Circ Res 2002 Apr. 19;90(7):764-9; and Horie, S. et al.,Biochem J 1992 Jan. 1;281 (Pt 1):149-54). Other vasoprotective effectsof ATRA include inhibition of endothelin-1, stimulation of plasminogenactivator synthesis, and increased beta 1 integrin expression (see, forexample, Yokota, J. et al. Atherosclerosis 2001 December;159(2):491-6;Kooistra,T. et al., Thromb Haemost 1991 May 6;65(5):565-72; and Medhora,M. M., Am J Physiol Heart Circ Physiol 2000 July;279(1):H382-H387). Whenprovided systemically or locally, in vivo, to balloon-injured rodentsand rabbits, ATRA has been shown to affect the vasculature, including,inhibition of neointimal hyperplasia, inhibition of vascular remodeling,accelerated reendothelialization, and prevention of restenosis inatherosclerotic rabbits. (See, for example, Miano, J. M. et al.,Circulation 1998 Sep. 22;98(12):1219-27; DeRose, Jr., J. J. et al.Cardiovasc Surg 1999 October;7(6):633-9; Lee, C. W. et al., J Korean MedSci 2000 February;15(1):31-6; Wiegman, P. J. et al., Arterioscler ThrombVasc Biol 2000 January;20(1):89-95; Herdeg, C. et al., Cardiovasc Res2003 February;57(2):544-53; Leville, C. D. et al., J Surg Res 2000 May15;90(2):183-90; and Leville, C. D. et al., Surgery 2000August;128(2):178-84.) Although encouraging, in vivo delivery of ATRAhas not been localized or sustained in these studies. More important, nostudies have been done with ATRA in the context of inhibiting neointimalhyperplasia in prosthetic grafts.

Implant Preparation

The preceding vascular implants can be prepared according to a methodcomprising, providing a vascular implant; and contacting the vascularimplant with all-trans retinoic acid (ATRA) to yield a modified vascularimplant. Generally, the modified vascular implant comprises abiocompatible polymeric matrix as described above and releases atherapeutically effective amount of ATRA sufficient to inhibitneointimal hyperplasia when implanted in a patient.

In certain embodiments, the vascular implant is a vascular graft, avascular stent, a wrap, or a gel. In a particular embodiment, thevascular implant is a vascular graft. In another particular embodiment,the vascular implant is a vascular stent. In another particularembodiment, the vascular implant is a wrap. In another particularembodiment, the vascular implant is a gel.

The vascular implant itself can be formed from biocompatible materialsknown to one skilled in the art. For example, vascular grafts can beformed from poly(ethylene terephthalate) (PETE, Dacron™) orpoly(tetrafluoroethylene), such as expanded poly(tetrafluoroethylene)(ePTFE). Vascular stents can be formed from stainless steel or acobalt-chromium alloy or nitnol.

In one embodiment, the contacting of the vascular implant with all-transretinoic acid (ATRA) can comprise coating the vascular implant with amixture comprising a biocompatible polymer prepolymer and nanoparticles,microparticles, micelles, or liposomes comprising ATRA. ATRA can readilybe incorporated into micro- and nanoparticles, micelles, and/orliposomes using a standard techniques known to those skilled in the art.The nanoparticles, microparticles, micelles, and/or liposomes can thenbe embedded within the biocompatible matrix of a coated vascular implantas noted above. In certain embodiment, the coated implant can be set,for example, by heating under vacuum to form a coherent film on thesurface of the implant comprising the biocompatible polymer andnanoparticles, microparticles, micelles, or liposomes comprising ATRA.When heating the implant, the temperature should be maintained at asuitable temperature which does not cause degradation of the ATRAembedded therein. For example, the implants can be heated at atemperature of about 40 to 60° C. for about 4 days.

The term “prepolymer” as used herein refers to a material which can beprocessed to form a coherent polymer film or matrix generally havingeither a higher molecular weight or higher crosslink density than theprepolymer. A prepolymer, for example, can be coated onto an objecteither as a liquid, oil, syrup, or from a solution and further processedto form a coherent polymer film or matrix by, for example, heating orcontacting the coated object with a polymerization catalyst. Examples ofprepolymers include, but are not limited to, polyester prepolymers. Forexample, a polyester prepolymer can be coated onto an object and set byheating to form a coherent polyester film or matrix having a highermolecular weight or higher crosslink density than the prepolymer.

In another embodiment, the contacting of the vascular implant withall-trans retinoic acid (ATRA) can comprise soaking the vascular implantin a solution comprising ATRA. Although ATRA is not readily soluble inwater, it is partly soluble in ethanol (3 mg/ml), and soluble indimethylsulfoxide (DMSO). It is also soluble in chloroform anddichloromethane, solvents commonly used to process biodegradablepolyester thermoplastics that are used in biomedical applications.

In another embodiment, the contacting of the vascular implant withall-trans retinoic acid (ATRA) can comprise coating the vascular implantwith a biocompatible polymer prepolymer; setting the coated vascularimplant; and soaking the coated vascular implant in a solutioncomprising ATRA. Setting the coated vascular implant can include heatingthe vascular implant, optionally under a static or dynamic vacuum, toencourage the film to cross-link or otherwise convert to a coherent filmwhich substantially coats the desired portion of a vascular implant. Incertain embodiments, when the coated vascular implant is soaked in asolution comprising ATRA, the solution causes the biocompatiblepolymeric matrix to swell.

In another embodiment, the contacting of the vascular implant withall-trans retinoic acid (ATRA) can comprise coating the vascular implantwith a mixture comprising ATRA and the biocompatible polymeric matrix,wherein the biocompatible polymeric matrix is a biocompatiblethermoplastic polymer.

In another embodiment, a wrap or gel comprising ATRA and thebiocompatible polymer can be placed around at least a portion of astandard vascular graft or stent for implantation in a patient in needthereof as described above. Such wraps and gels can be prepared asdescribed above. For example, a prepolymer, as described above, can bepolymerized to provide a wrap or gel which can be contacted with asolution comprising ATRA to provide an ATRA containing wrap or gel.Alternatively, a prepolymer comprising ATRA can be can be polymerized toprovide to provide an ATRA containing wrap or gel. The wrap or gel canbe contacted with a solution comprising ATRA prior to being placedaround at least a portion of a vascular graft or stent; oralternatively, the wrap or gel can be placed around at least a portionof a vascular graft or stent, and then the coated graft or stent can becontacted with a solution comprising ATRA.

EXAMPLES Example 1 Poly(1,8 octanediol-co-citrate)(POC) Pre-PolymerSynthesis

Equimolar amounts of citric acid and 1,8-octanediol are melted togetherat 160° C. while stirring for 15 minutes The temperature can besubsequently decreased to 140° C. and the mixture stirred for 1 hour.The pre-polymer can then be purified by dissolution in ethanol followedby precipitation in water and freeze-dried. For surface modification ofan ePTFE graft, POC pre-polymer is dissolved in ethanol or 1,3-dioxolaneto a concentration of 10% (w/v). See, for example, Yang J, Webb A R,Pickerill S J, Hageman G, Ameer G A. Synthesis and evaluation ofpoly(diol citrate) biodegradable elastomers. Biomaterials 2006 Mar;27(9):1889-98; and Yang J, Webb A R, Ameer G A. Novel citric acid-basedbiodegradable elastomers for tissue engineering. Adv Mater2004;16(6):511-6, each of which are hereby incorporated by reference intheir entirety).

The mechanical properties of the preceding elastomer can be modulated bycontrolling synthesis conditions such as crosslinking temperature andtime, vacuum, choice of diol, and initial monomer molar ratio. Young'smodulus can range from 1 to 16 MPa, with strengths and strains at breakof up to 10 MPa and 500%, respectively. Depending on the synthesisconditions, total degradation time may range from months to years.

Increasing the molar ratio of citric acid increases the degradation rateof the copolymer without sacrificing its initial tensile strength.Likewise, degradation can be modulated by including glycerol (2.5 mole%). Degradation of this glycerol-containing elastomer in phosphatebuffered saline (PBS) at 37° C. was enhanced almost 2-fold after 4months of incubation.

Example 2 In Vitro Evaluation of Clotting and InflammatoryCharacteristics of POC

Re-calcification clotting assays were performed to assess the clottingkinetics of POC relative to tissue culture polystyrene and PLGA.Briefly, test and control polymer samples in 96-well plates wereincubated in acid citrate dextrose (ACD) anticoagulated human or pigplatelet poor plasma (PPP). Immediately prior to absorbance measurement,CaCl₂ (0.025 M) was added to each well to initiate clotting. Theabsorbance of each well was monitored every 30 seconds for 30 minutes at405 nm. The rate of clot formation is consistently lower for plasmaexposed to POC when compared to the other materials (slope of linearregion=0.088±0.027, 0.089±0.013, and 0.025±0.019 A.U./min, for TCP,PLGA, and POC, respectively). This finding may be explained by thepresence of the hydroxyl, carboxyl, and potentially chelating citricacid functional groups within the POC.

For assessment of inflammatory potential, a suspension of humanmonocytic THP-1 cells was exposed to POC, TCP, PLGA, and ePTFE films.Afterwards, the expression of tissue factor, IL-6, and TNF-α wasmeasured via ELISA. Addition of lipopolysaccharide (LPS) to the cellswas used as a positive control. Results are shown in Table 1.

TABLE 1 TCP ePTFE PLGA POC LPS Tissue 208 ± 26  96 ± 27 114 ± 12  9 ± 2*536 ± 81  factor TNF-α 12 ± 2 21 ± 3 15 ± 5 17 ± 5  874 ± 531 IL-6  1.4± 0.05 32 ± 5  8 ± 4 2.8 ± 2*  —POC did not elicit a significant upregulation of these markers relativeto the other materials. In fact, POC was less reactive than ePTFEregarding tissue factor and IL-6 expression.

Example 3 Modification of ePTFE Grafts with POC

Prior to modification, standard-wall non-stretch ePTFE grafts werecleaned by first soaking under sonication in absolute ethanol, acetone,and vacuum drying. The lumen of ePTFE grafts were modified bymechanically coating a layer of POC through a spin-shearing method.Briefly, a 5 mm diameter glass rod was dipped into 10% POC pre-polymer(pre-POC, Example 1) solution in 1,4-dioxane and inserted horizontallyinto the motor of a mechanical stirrer. The pre-POC-coated glass rod wasspun clockwise at 300 rpm for 2 minutes and a 6 cm-long piece of ePTFEgraft was placed concentrically over the spinning rod. The lumen of thegraft was sheared against the spinning rod for 2 minutes by manuallyrotating the graft counterclockwise. The above procedure was consideredto be 1 coating. To change the amount of POC deposited onto the graft(and, therefore, the coating thickness), the above procedure wasrepeated 3 and 6 times (defined as 3 and 6 coatings) to assess POCcoverage and effects on graft compliance with increasing polymercontent. After air-drying, the pre-POC-coated ePTFE graft was put intoan oven at 80° C. for 2 days to obtain POC-ePTFE grafts.

For characterization, samples were cut into 1 cm² pieces. Changes insurface characteristics of the POC-modified ePTFE samples were assessedvia SEM, water-in-air contact angle measurement, Fourier transforminfrared (FTIR) analysis, and x-ray photoelectron spectroscopy (XPS)analysis. The compliance of the modified grafts was also measured.Sampling from several sections of the grafts confirmed uniform coatingsof the PTFE fibrils and nodes. The thickness of the POC coating isapproximately 2-5 microns. Equilibrium water-in-air contact angles ofPOC-ePTFE versus unmodified ePTFE were 36° and 120° , respectively. FTIRand XPS confirmed the presence of carboxyl and hydroxyl groups withinthe lumen of the graft. Three coatings or treatments with POC did nothave an effect on the compliance of the native graft. The small decreasein compliance with 6 coatings may be explained by a severe disruption ofthe graft's fibril-node microarchitecture.

Example 4 Surface Modification of ePTFE Graft with POC and Drug Loading

Prior to modification, standard-wall non-stretch ePTFE grafts arecleaned by first soaking under sonication in absolute ethanol and vacuumdrying. A POC infusion method is used in order to ensure a large amountof POC available for drug loading. POC is infused through the vesselwall by clamping one end of the graft and pumping the POC pre-polymersolution (Example 1) into the graft and through the vessel wall. Afterinfusing polymer through the graft wall and drying, the lumen of ePTFEgrafts is modified by mechanically coating a layer of POC through aspin-shearing method.

A 5 mm diameter glass rod is dipped into 10% POC pre-polymer solution in1,3-dioxolane and inserted horizontally into the motor of a mechanicalstirrer. The pre-POC-coated glass rod is spun clockwise at 300 rpm for 2minutes and an 8 cm-long piece of ePTFE graft placed concentrically overthe spinning rod. The lumen of the graft is sheared against the spinningrod for 2 minutes by manually rotating the graft counterclockwise. Theabove procedure is considered to be 1 coating. A total of 3 coatings canbe applied to the graft as it has been shown that up to 3 coatings canbe applied without significantly affecting compliance. The coating isuniform and can remain intact after one month of implantation in vivo.After air-drying, the pre-POC-coated ePTFE graft is placed into an ovenfor post-polymerization.

Dosing of retinoic acid (ATRA) is controlled through the drug releaserate which can be controlled by changing the POC polymerizationconditions to vary the degree of swelling in aqueous media. For a fasterrelease rate, coated grafts are polymerized at 80° C. for 2 days. Slowerreleasing grafts are polymerized for an additional 2 days at 80° C.Alternatively, 1,12 dodecanediol can be added to confer hydrophobicityto the coating. ATRA is loaded and sterilized at the same time bysoaking the coated graft in a retinoic acid solution in ethanol (3mg/m1) at room temperature in the dark for 24 hours. After removal fromthe ATRA solution, the grafts are freeze dried in sterile containers tocollapse the polymer and entrap the ATRA. The amount of ATRA loaded inthe polymer film can be indirectly determined by measuring theconcentration of ATRA remaining in the ethanol solution after soaking.The concentration of ATRA in solution can be determinedspectrophotometrically at 350 nm and compared to a standard curve.

Example 5 ATRA Release Kinetics and POC Stability

To measure the release, isomerization, and degradation of retinoic acidfrom POC, disks (10 mm diameter, 1 mm thick) and small segments ofATRA-loaded POC-ePTFE graft (1 cm length, 6 mm diameter) can be placedin culture medium and the drug release and POC degradation monitoredover a period of 6 months. POC degradation can be assessed via mass lossand SEM. The supernatant can be removed from the disk or graft segmentand replaced with fresh medium. Briefly, 350 μL can be taken from themedium at various time points and 50 μL of 1 M sodium acetate and 600 μLof acetonitrile can be added. After vortexing and centrifuging, 720 μLof the supernatant can be placed in a 2 mL glass autosampler vial alongwith 240 μL of water. After inversion mixing, the vial can be placed inthe autosampler at 4° C. and the drug content determined usingreverse-phase HPLC with UV-Vis detector at 340 nm.

Retinoids can be identified using external standards for4-oxo-trans-retinoic acid, 4-oxo-cis-retinoic acid, 13-cis-retinoicacid, and ATRA. Supernatants can also be assessed for ATRA activity.Non-drug loaded POC-ePTFE grafts and uncoated ePTFE grafts can be usedas controls. Also, ATRA-loaded POC-ePTFE grafts (9 cm length, 6 mmdiameter) can be placed in a pulsatile flow circuit (1 Hz) to mimicdynamic conditions found in vivo. Cell culture medium can be perfused,single pass at 200 and 300 mL/min (flow rates typical of carotid andmedium size arteries) and samples can be collected downstream from thegraft. Samples can be assessed for ATRA concentration and activity (andPOC fragmentation). These experiments can provide some insight into ATRAconcentrations in the bulk flow at the distal anastomosis and effects onendothelial and smooth muscle cells.

Example 6 Cellular Responses of Released ATRA

The activity of the released ATRA can be assessed using both porcine andhuman aortic smooth muscle and endothelial cells as previouslydescribed. Human aortic smooth muscle (HASMC) and endothelial cells(HAEC) can be purchased from Lonza (Lonza, Allendale, N.J.). Porcineaortic smooth muscle (PASMC) and endothelial cells (PAEC) can bepurchased from Cell Applications Inc. (Cell Applications, Inc., SanDiego, Calif.). Samples collected from the static and flow drug releasestudies can be directly added to the wells to assess the effect ofreleased ATRA on cells (i.e. ATRA activity). Positive controls canconsist of adding ATRA-dissolved in DMSO or ethanol to the cells. Theseexperiments can be also conducted with ATRA-loaded POC-ePTFE segmentsplaced directly in the wells (1 cm length, 6 mm diameter).

Cell Proliferation: HASMC, PASMC, HAEC, and PAEC can be seeded on 6-wellplates at a density of 5000 cells/cm². The next day, the baseline numberof intact cells can be determined using a Picogreen DNA assay kit(Invitrogen, Carlsbad, Calif.) after trypsinizing and pelleting thecells. The graft segment can then be placed above the cells in atranswell insert and at various time points the number of cells can bedetermined using a Picogreen DNA assay.

Cell Migration: The migration of smooth muscle cells can be examinedalone and in the presence of endothelial cells. To test the migrationwithout endothelial cells, smooth muscle cells can be seeded at adensity of 5000 cells/cm² in 6-well plates. After the cells reachconfluence, the culture can be scraped with a silicon-coated stick toobtain a 0.8 mm wide in vitro wound and photographed using a Nikonmicroscope (Nikon Eclipse, TE2000-U) equipped with a photometricsCoolSNAP HQ (Silver Spring, MD). A graft segment can then be placed ineach well. After 24 hours, the graft can be removed and cells can bestained with propidium iodide to count migrating cells invading theempty space. At least 6 images from the empty space can be used andresults counted as the total number of migrating cells per field. Toexamine smooth muscle cell migration in the presence of endothelialcells, a transfilter system can be prepared as previously described.Briefly, Nucleopore polycarbonate filters (5 μm pore size) can beinserted between an inner and outer polycarbonate frame. In this way,two separate compartments are created in which endothelial cells andsmooth muscle cells can be seeded. The 5 μm pore size can allow themigration of smooth muscle cells from the upper to the lower chamber asshown previously. Endothelial cells can be seeded at a density of 5000cells/cm² on the lower filter side. After 24 hours, smooth muscle cellscan be seeded on the upper filter side and a graft segment added to thelower compartment. After 14 days in culture, the total number of cellson both sides of the filter can be determined using a Picogreen DNAassay after disaggregation with trypsin/EDTA. Cell proliferation andmigration can be compared among all experimental groups. As shown inprevious experiments, endothelial cells should stop proliferating onceconfluence is reached, therefore any increase in the number of cellsshould be due to smooth muscle cell proliferation and migration.

Nitric Oxide Release: Endothelial cells can be examined for nitric oxiderelease in response to ATRA. Intracellular nitric oxide can be examinedusing a nitric oxide synthase detection kit according to manufacturer'sinstructions (Cell Technology Inc, Mountain View, Calif.). Nitric oxideproduction can be determined by measuring fluorescence intensity withexcitation at 495 nm and emission at 510 nm. Extracellular nitric oxideproduction can be assessed using a nitric oxide analyzer (Apollo 4000,World Precision Instruments, Sarasota, Fla.).

Protein Expression and Synthesis: Protein expression and synthesis canbe measured concurrently with cell proliferation. Smooth muscle cellscan be probed for smooth muscle α-actin and heavy chain myosin viaimmunohistochemistry and western blotting to assess differentiation. Theability of the cells to remodel the extracellular matrix can be assessedby staining for matrix metalloproteinase-2 and matrixmetalloproteinase-9. Detection can then be performed using horseradishperoxidase-linked secondary antibodies and chemiluminescence detectionvia a UVP Biochemi gel documentation system (UVP, Inc, Upland, Calif.).Collagen and elastin synthesis can be determined using the Sircolcollagen and Fastin elastin assay kits (Accurate Chemical Co., Westbury,N.Y.).

Example 7 Surgical Implantation and In vivo Graft Assessment

Grafts can be sterilized during the loading step by soaking in retinoicacid in ethanol and placed in sterile containers for freeze drying.Conventional pigs can be fasted overnight prior to the day of surgerybut allowed ad libitum access to tap water. The animals can receivepre-op analgesia with buprenorphine (0.01 mg/kg IM), and sedation withAcepromazine (0.15 mg/kg IM) and Ketamine (20 mg/kg IM). Afterintubation, maintenance anesthesia can be conducted with Isoflurane(0.5-2.0%) delivered with 100% oxygen.

Procedure #1 (carotid artery bypass graft): Male pigs (30-35 kg) canundergo vascular graft implantation in the carotid arteries. Bilateralcommon carotid arteries (CCAs) can be exposed through a midline neckincision. Five minutes before CCA occlusion, heparin (150 U/kg) can beadministered intravenously. A 6 cm long segment of the graft (6 mmthin-walled non-stretch non-ringed ePTFE, POC-coated and either drugloaded or untreated) can be anastomosed to the proximal and distal CCAin a standard end-to-side configuration using 6-0 polypropylene stures.The carotid artery can be ligated to simulate an arterial occlusion.Prior to completion of the anastomosis, the vessel can be back-bled andflushed with heparinized saline. After completion of the anastomoses,flow can be restored and manually confirmed with palpation. The neckincisions can be closed with absorbable suture. Heparin can not beadministered after surgery. Postoperative analgesia can be provided withbuprenex (0.005-0.01 mg/kg IM) every 12 hours×48 hours. This procedurecan be conducted on the left and right CCA, as two grafts can beimplanted per animal. Aspirin (325 mg daily) can be given as anantiplatelet therapy pre- and post-operatively.

Procedure #2 (2D contrast angiography): Angiograms can be performed viaa 6F sheath inserted into the right common femoral artery percutaneouslyand advanced into the proximal CCA under fluoroscopic guidance.Selective angiograms of both carotid artery bypass grafts can beobtained using 10 cc of a non-iodinated contrast agent (visipaque oromnipaque). Following completion of the angiogram, the guidewire andcatheter can be removed.

Procedure #3 (MRA): In lieu of contrast angiography, grafts may beassessed for patency and flow using Magnetic Resonance Imaging Analysis(MRA). The animals can undergo general anesthesia as described above,and receive a gadolinium-based contrast agent intravenously in order toobtain MRA images.

Procedure #4 (ultrasonography): Grafts also can be evaluated by duplexultrasonography prior to harvesting to assess patency and obtainvelocity measurements [peak systolic (PSV) and end diastolic velocity(EDV)] throughout the graft to evaluate for areas of stenoses. Asignificant stenosis can be defined as a PSV greater than two times thenormal inflow artery velocity. Following harvest of the grafts, theanimal can undergo euthanasia via pentobarbital overdose and bilateralthoracotomy.

Example 8 Graft Processing and Analysis

The grafts and adjacent 3 cm segments of the native vessel at eachanastomosis can be harvested and cut into two parts from the middle(distal and proximal specimens). In select animals, a section of thegraft can be opened longitudinally, photographed, and assessed forpercent thrombus-free surface area. A 0.5 cm-long section of the graftfrom the distal and proximal specimens can be fixed in a 2.5%glutaraldehyde solution for morphological assessment via SEM The rest ofthe specimen can be fixed in 10% neutral buffered formalin, embedded inparaffin, and cut into 5 micron sections.

All grafts can be assessed for: a) neointimal hyperplasia (H&E), b)cellular proliferation (H&E stain, anti-Ki67) and differentiation (aactin and heavy chain myosin); b) collagen and elastin (Masson'strichrome and modified elastin van Gieson stain, respectively); c)presence of endothelial cells (von Willebrand and VE-cadherin); and d)inflammation (MAC387 antibody for macrophages, anti-CD45 antibody forleukocytes). Segments of all grafts can also undergo SEM formorphological assessment of any platelet or white celladhesion/activation and the presence/absence of endothelial cells andPOC.

Histomorphometric analysis: To quantify the degree of neointimalhyperplasia, each anastomosis can be sectioned in entirety and 5equally-spaced sections throughout each anastomosis can be assessed toquantify neointimal hyperplasia. Each section can be imaged andneointimal area, medial area, luminal area, and circumference can bemeasured using ImageJ software (NIH). To quantify cellularproliferation, inflammation, and endothelialization, nuclei ofpositively staining cells can be counted in four different high powerfields per section. Since macrophages appear mostly at the lumen/graftinterface, the interface can be taken as the reference point. Theinterface plus 250 μm into the lumen and 250 μm into the graft's wallcan define the area of interest. All the evaluations can be performed ina blinded manner to maintain an objective interpretation of the results.The data obtained from the POC-based grafts and control ePTFE graftsplaced in each pig can be compared to each other. Statisticalcomparisons can be performed using the student's t-test.

Patency: Patency and degree of stenosis of the grafts can be assessednoninvasively via MRA or contrast angiography, and duplexultrasonography prior to euthanasia and graft harvest.

1.-20. (canceled)
 21. A method for reducing the occurrence of neointimalhyperplasia and/or thrombosis following implantation of a vascularimplant, said method comprising, contacting a vascular implant withall-trans retinoic acid (ATRA); and implanting the vascular implant in apatient in need of thereof, wherein the vascular implant comprises abiocompatible polymeric matrix selected from the group consisting ofpoly(citric acid-diol) or poly(glycerol-diacid), polyurethane,polycarbonate, polyanhydride, polyphosphoester, and mixtures thereof;and the vascular implant locally releases a therapeutically effectiveamount of ATRA sufficient to inhibit neointimal hyperplasia and/orthrombosis by diffusing from the vascular implant to a blood vesselsurface adjacent to the vascular implant when implanted in the patient.22. The method of claim 21, wherein the vascular implant is a vasculargraft, a vascular stent, or a wrap, or a gel.
 23. The method of claim21, wherein the vascular implant comprises a vascular stent or graft anda wrap or gel, wherein the wrap or gel comprises the biocompatiblepolymeric matrix and ATRA, and wherein the wrap or gel is placed aroundat least a portion of the vascular stent or graft.
 24. The method ofclaim 21, wherein the vascular implant releases ATRA at a rate of about0.001 to 5 mg per gram of biocompatible polymeric matrix per day. 25.The method of claim 21, wherein the vascular implant comprises about0.001 to 15 wt % ATRA with respect to the biocompatible polymericmatrix.
 26. The method of claim 21, wherein the therapeuticallyeffective amount of ATRA is released for a period of about 1 day toabout 12 weeks.
 27. The method of claim 21, wherein the biocompatiblepolymeric matrix is a poly(citric acid-diol) or poly(glycerol-diacid).28. The method of claim 21, wherein the biocompatible polymeric matrixis elastomeric or a gel.
 29. The method of claim 21, whereinmicroparticles or nanoparticles comprise the ATRA, wherein themicroparticles or nanoparticles are embedded within the biocompatiblepolymeric matrix.
 30. The method of claim 21, wherein the ATRA isencapsulated in micelles or liposomes, wherein the micelles or liposomesare embedded within the biocompatible polymeric matrix.
 31. The methodof claim 21, wherein the ATRA is absorbed within the biocompatiblepolymeric matrix.
 32. The method of claim 21, wherein the ATRA issuspended within or dissolved within the biocompatible polymeric matrix.33. A method for preparing a modified vascular implant, comprisingproviding a vascular implant; and contacting the vascular implant withall-trans retinoic acid (ATRA) to yield a modified vascular implant,wherein the modified vascular implant comprises a biocompatiblepolymeric matrix selected from the group consisting of poly(citricacid-diol) or poly(glycerol-diacid), polyurethane, polycarbonate,polyanhydride, polyphosphoester, and mixtures thereof; and the modifiedvascular implant releases a therapeutically effective amount of ATRAsufficient to inhibit neointimal hyperplasia and/or thrombosis whenimplanted in a patient.
 34. The method of claim 33, wherein thecontacting comprises coating the vascular implant with a mixturecomprising a biocompatible polymer prepolymer and nanoparticles,microparticles, micelles, or liposomes comprising ATRA.
 35. The methodof claim 34, wherein the biocompatible polymer prepolymer is a polyesterprepolymer.
 36. The method of claim 33, wherein the contacting comprisessoaking the vascular implant in a solution comprising ATRA.
 37. Themethod of claim 33, wherein the contacting comprises coating thevascular implant with a biocompatible polymer prepolymer; setting thecoated vascular implant; and soaking the coated vascular implant in asolution comprising ATRA.
 38. The method of claim 37, wherein thesolution causes the biocompatible polymeric matrix to swell.
 39. Themethod of claim 33, wherein the contacting comprises coating thevascular implant with a mixture comprising ATRA and the biocompatiblepolymeric matrix, wherein the biocompatible polymeric matrix is abiocompatible thermoplastic polymer.